PROJECT 3 SUMMARY (Manipulation of sperm-specific proteases using genetic and chemical approaches) The overall goals of Project 3 are to elucidate the molecular mechanisms of action of male reproductive tract-specific serine protease-like enzymes through the use of genetics and chemical biology, and to use these data to guide discovery of novel non-hormonal contraceptive agents. Biological targets ideal for drug discovery endeavors are those with significant and non-redundant biological effects, highly specific molecular functions, and biochemical features amenable to molecular inhibition. No single definitive methodology exists for the identification or validation of efficacious drug targets, and certainly the processes of evolution are not driven to yield numerous physiologically vulnerable pathways, particularly for contraception. Therefore, a thorough understanding of the molecular mechanisms governing fertility as well as an approach that samples a wide array of intervention points is desirable. As a complement to targets emerging from Projects 1 and 2, Project 3 will focus on the genetic and chemical analyses of five serine protease-like enzymes with expression limited to the male reproductive tract. These mechanistic studies will allow us to place these serine protease-like enzymes into reproductive pathways and simultaneously determine the utility of each of these proteins as a contraceptive target. Early studies of the sperm-zona pellucida interaction assumed that enzymes contained within the sperm acrosome were required for penetration of cumulus cell layers and the zona pellucida. More recently, it has become apparent that whereas the exocytotic event associated with release of acrosomal enzymes (during the acrosome reaction) is essential for fertilization, it is neither induced by contact with the zona pellucida, nor required to occur in proximity of the outer vestments of the oocyte. Similarly, motile sperm are essential for successful fertilization. Our overall hypothesis is that serine protease-like enzymes are a novel class of proteins required for male fertility and the discovery of small-molecule inhibitors for these spermatogenic-required enzymes will lead to the development of unique male contraceptives. Our Specific Aims are as follows: 1) Use CRISPR/Cas9 models of male reproductive tract-specific proteases to clarify their requirement in reproduction; 2) Aid in the expression and purification of recombinant proteins from Projects 1, 2, and 3 for DNA-encoded chemistry technology (DEC- Tec) affinity selections; and 3) Use DEC-Tec to uncover small-molecule probes and inhibitors of novel serine protease-like enzymes required for fertility and evaluate early drug-like leads and analogues with acceptable pharmacokinetic properties in proof-of-concept contraceptive studies in vivo.